U.S. patent number 9,285,314 [Application Number 14/350,896] was granted by the patent office on 2016-03-15 for systems and methods enabling high-throughput, real time detection of analytes.
This patent grant is currently assigned to Brown University. The grantee listed for this patent is Brown University. Invention is credited to Jing Feng, Henri J. Lezec, Vihang Mehta, Domenico Pacifici, Tayhas G. r. Palmore, Steve Rhieu, Alec Roelke, Vince Siu.
United States Patent |
9,285,314 |
Pacifici , et al. |
March 15, 2016 |
Systems and methods enabling high-throughput, real time detection
of analytes
Abstract
System and methods for detecting analytes are provided. The
system includes a plasmonic interferometer with a first surface
having a first and second scattering element and an aperture
disposed between the first scattering element and the second
scattering element. A first distance between the aperture and the
first scattering element and a second distance between the aperture
and the second scattering element are selected to provide
interference of light at the slit. The system also includes a light
source for illuminating the first surface of the plasmonic
interferometer, a detector positioned for detecting light
transmitted through the aperture, and a sample holder for disposing
a sample to be analyzed onto the first surface of the plasmonic
interferometer.
Inventors: |
Pacifici; Domenico (Providence,
RI), Lezec; Henri J. (Bethesda, MD), Palmore; Tayhas G.
r. (Providence, RI), Siu; Vince (Thornhill,
CA), Mehta; Vihang (Pune, IN), Roelke;
Alec (Basking Ridge, NJ), Rhieu; Steve (Rockville,
MD), Feng; Jing (Jiangsu, CN) |
Applicant: |
Name |
City |
State |
Country |
Type |
Brown University |
Providence |
RI |
US |
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Assignee: |
Brown University (Providence,
RI)
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Family
ID: |
48082526 |
Appl.
No.: |
14/350,896 |
Filed: |
October 12, 2012 |
PCT
Filed: |
October 12, 2012 |
PCT No.: |
PCT/US2012/060079 |
371(c)(1),(2),(4) Date: |
April 10, 2014 |
PCT
Pub. No.: |
WO2013/056137 |
PCT
Pub. Date: |
April 18, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140327913 A1 |
Nov 6, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61662048 |
Jun 20, 2012 |
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61581951 |
Dec 30, 2011 |
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61546435 |
Oct 12, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
21/45 (20130101); G01N 21/553 (20130101); G01N
21/554 (20130101) |
Current International
Class: |
G01B
9/02 (20060101); G01N 21/45 (20060101); G01N
21/552 (20140101) |
Field of
Search: |
;356/450 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chan et al., "Transmission Enhancement in an Array of Subwavelength
Slits in Aluminum Due to Surface Plasmon Resonances," Bell Labs
Technical Journal, vol. 10, No. 3, No Month Listed 2005 (pp.
143-150). cited by applicant .
Genet et al., "Light in Tiny Holes," Nature, vol. 445, Jan. 4, 2007
(pp. 39-46). cited by applicant .
International Search Report and Written Opinion Issued by the U.S.
Patent and Trademark Office as International Searching Authority
for International Application No. PCT/US2012/60079 mailed Jan. 9,
2013 (11 pgs). cited by applicant .
Lee et al., "Sensitive Biosensor Array Using Surface Plasmon
Resonance on Metallic Nanoslits," Journal of Biomedical Optics,
vol. 12, No. 4, Jul./Aug. 2007 (pp. 044023-1-044023-5). cited by
applicant .
Leong et al., "Surface Plasmon Resonance in Nanostructured Metal
Films under the Kretschmann Configuration," Journal of Applied
Physics, vol. 106, No Month Listed 2009 (pp. 124314-1-124314-5).
cited by applicant .
Perney et al., "Tuning Localized Plasmons in Nanostructured
Substrates for Surface-Enhanced Raman Scattering," Optical Society
of America, No Month Listed 2005 (11 pages). cited by applicant
.
Tanemura et al., "Multiple-Wavelength Focusing of Surface Plasmons
with a Nonperiodic Nanoslit Coupler," Nano Letters, vol. 11, No
Month Listed 2011 (pp. 2693-2698). cited by applicant .
Zayats et al., "Nano-Optics of Surface Plasmon Polaritons," Physics
Reports, vol. 408, No Month Listed 2005 (pp. 131-314). cited by
applicant.
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Primary Examiner: Chowdhury; Tarifur
Assistant Examiner: Rahman; MD
Attorney, Agent or Firm: Wilmer Cutler Pickering Hale and
Dorr LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a national stage application of International
Application No. PCT/US12/60079, filed on Oct. 12, 2012, entitled
"SYSTEMS AND METHODS ENABLING HIGH-THROUGHPUT, REAL TIME DETECTION
OF ANALYTES," incorporated herein by reference in its entirety,
which claims priority under 35 U.S.C. .sctn.119(e) to U.S.
Provisional Application No. 61/662,048, filed on Jun. 20, 2012,
entitled "ENHANCING THE SENSITIVITY AND SELECTIVITY OF PLASMONIC
INTERFEROMETERS USING DYE CHEMISTRY," which is incorporated herein
by reference in its entirety which is incorporated herein by
reference in its entirety. This application also claims priority
under 35 U.S.C. .sctn.119(e) to U.S. Provisional Application No.
61/581,951, filed on Dec. 30, 2011, entitled "NANOSCALE PLASMONIC
INTERFEROMETERS FOR MULTISPECTRAL, HIGH-THROUGHPUT BIOCHEMICAL
SENSING," which is incorporated herein by reference in its
entirety. This application also claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 61/546,435, filed
on Oct. 12, 2011, entitled "SYSTEMS AND METHODS ENABLING
HIGH-THROUGHPUT, REAL TIME DETECTION OF BIOCHEMICAL ANALYTES,"
which is incorporated herein by reference in its entirety.
Claims
What is claimed is:
1. A system for detecting an analyte comprising: a plasmonic
interferometer, the plasmonic interferometer comprising a first
surface and a second surface on an opposite side of the plasmonic
interferometer than the first surface, the first surface having a
first groove and a second groove and a slit disposed between the
first groove and the second groove to define a first distance
between the slit and the first groove and a second distance between
the slit and the second groove, wherein the first and second
distances are selected to provide interference of light at the
slit; a light source for illuminating the first surface of the
plasmonic interferometer; a detector for detecting light
transmitted through the slit, the detector positioned proximate to
the second surface of the plasmonic interferometer; and a sample
holder for disposing a sample to be analyzed onto the first surface
of the plasmonic interferometer.
2. The system of claim 1, wherein the first and second scattering
elements are surface plasmon polariton scattering elements.
3. The system of claim 1, wherein the aperture slit is designed to
allow collection of a light property.
4. The system of claim 1, wherein the plasmonic interferometer
comprises a conductive film.
5. The system of claim 4, wherein the plasmonic interferometer
comprises one of gold (Au), silver (Ag), aluminum (Al), copper
(Cu), or other noble metal, and a functional protective layer such
as indium-tin oxide (ITO), or silicon dioxide, or other functional
material.
6. The system of claim 1, wherein the sample holder comprises walls
positioned to retain the sample on the first surface of the
plasmonic interferometer.
7. The system of claim 1, wherein the sample holder comprises a
microfluidic channel positioned to direct a sample over the first
surface of the plasmonic interferometer.
8. The system of claim 1, wherein the first surface of the
plasmonic interferometer is functionalized with an analyte capture
agent selected to capture the analyte to be detected.
9. The system of claim 1, wherein the first surface of the
plasmonic interferometer is coated with a dielectric material.
10. The system of claim 1, wherein the first surface of the
plasmonic interferometer is coated with an analyte capture agent
selected to capture the analyte to be detected.
11. The system of claim 1, wherein the analyte capture agent is
selected to capture cytokines.
12. The system of claim 1, further comprising a power source.
13. The system of claim 1, wherein the detector is a CCD
camera.
14. The system of claim 1, wherein the detector is a CMOS
camera.
15. The system of claim 1, wherein the detector is external.
16. The system of claim 1, wherein the detector is internal.
17. The system of claim 1, wherein the first scattering element and
the second scattering element have a length between 10 nm and 100
.mu.m.
18. The system of claim 1, wherein the first scattering element and
the second scattering element have a width between 10 nm and 500
nm.
19. The system of claim 1, wherein the first and the second
scattering element have a depth between 1 nm and 300 nm.
20. The system of claim 1, wherein the aperture has a width between
50 nm and 500 nm.
21. The system of claim 1, wherein the second distance is greater
than the first distance.
22. The system of claim 1, wherein the second distance is equal to
the first distance.
23. The system of claim 1, wherein the light source is selected to
provide light at optical frequencies.
24. The system of claim 1, further comprising a protective layer of
a dielectric material, or a functional layer, or oxidative dye, or
chromogenic dye disposed on the first surface.
25. The system of claim 1, wherein the first scattering element and
the second scattering element are linear.
26. The system of claim 1, wherein the first scattering element and
the second scattering element are arcs.
27. The system of claim 1, wherein the aperture is a hole.
28. The system of claim 1, wherein the first scattering element and
the second scattering elements are curvilinear.
29. The system of claim 1, further comprising inlet and outlets
positioned to permit inflow and outflow of a second fluid in the
sample holder.
30. The system of claim 1, wherein the first and second scattering
elements comprise a plurality of scattering elements of different
sizes, shapes and/or dimensions, and the aperture comprises several
slits of different sizes, shapes and/or dimensions.
31. A method of real-time detection of an analyte comprising:
providing a plasmonic interferometer according to claim 1 applying
a sample to be analyzed on the first surface of the plasmonic
interferometer; illuminating the plasmonic interferometer with the
light source; measuring a light property of a composite light
transmitted through the aperture; and determining a characteristic
of an analyte of interest based on the measured light intensity;
wherein the composite light is generated through interference at
the aperture of the incident light and scattered light from the
first and second scattering elements.
32. The method of claim 31, wherein the step of measuring a light
property includes measuring a light intensity of the composite
light transmitted through the aperture.
33. The method of claim 31, wherein a first wave of scattered light
propagates from the first scattering element to the aperture; a
second wave propagates from the second scattering element to the
aperture; and wherein the first and second distances are selected
to provide constructive interference of the first and second
light.
34. A method of real-time detection of an analyte comprising:
providing a plasmonic interferometer according to claim 1; applying
a sample to be analyzed on the first surface of the plasmonic
interferometer, wherein the sample comprises an oxidative or
chromogenic dye selected to absorb particular light frequencies;
illuminating the plasmonic interferometer with the light source;
measuring a light property of a composite light transmitted through
the aperture; and determining a characteristic of an analyte of
interest based on the measured light intensity; wherein the
composite light is generated through interference at the slit of
the incident light and scattered light from the first and second
scattering elements.
35. A method of spectroscopic measurements of the dispersion
relation and optical constants of dielectric materials in gaseous,
liquid, or solid form, the method comprising: providing a plasmonic
interferometer according to claim 1; applying a sample to be
analyzed on the first surface of the plasmonic interferometer,
wherein the sample comprises at least one of a dielectric material
and a mixture of dielectric material; illuminating the plasmonic
interferometer with a light source; measuring a light property of a
composite light transmitted through the aperture; and determining a
characteristic of an analyte in the sample based on the measured
light property; wherein the composite light is generated at the
aperture through interference of incident light from the source and
scattered light from the first and second scattering elements.
36. A system for detecting a plurality of analytes comprising: a
plurality of plasmonic interferometers, each plasmonic
interferometer comprising: a first surface and a second surface on
an opposite side of the plasmonic interferometer than the first
surface, the first surface having a first groove and a second
groove; and a slit disposed between the first groove and the second
groove to define a first distance between the slit and the first
groove and a second distance between the slit and the second
groove; wherein the first and second distances are selected to
provide controlled interference of light at the aperture slit; a
light source for illuminating the first surface of each of the
plurality of plasmonic interferometers; a plurality of detectors
for detecting light transmitted through the slit of each plasmonic
interferometer, each detector positioned proximate to the second
surface of a corresponding plasmonic interferometer; and a
plurality of sample holders, each sample holder disposing a sample
to be analyzed onto the first surface of each plasmonic
interferometer.
37. The system of claim 36, wherein the number of plasmonic
interferometers varies between 1 and 10,000,000,000.
38. The system of claim 36, wherein the plasmonic interferometers
are etched over an area that varies between 1E-6 mm.sup.2 and 1E4
mm.sup.2.
39. The system of claim 36, wherein two neighboring plasmonic
interferometers are separated by a distance of at least two
propagation lengths.
40. The system of claim 36, wherein two neighboring plasmonic
interferometers are separated by a thin film of platinum or
chromium.
41. The system of claim 36, wherein neighboring plasmonic
interferometers are optimally staggered to minimize cross-talk
along the plane.
42. A system for detecting an analyte comprising: a plasmonic
interferometer, the plasmonic interferometer comprising a first
surface and a second surface on an opposite side of the plasmonic
interferometer than the first surface, the first surface having a
first groove and a second groove and a slit disposed between the
first groove and the second groove to define a first distance
between the slit and the first groove and a second distance between
the slit and the second groove, wherein the first and second
distances are selected to provide interference of light at the
slit; a light source for illuminating the first surface of the
plasmonic interferometer; a detector for detecting light
transmitted through the slit, the detector positioned proximate to
the second surface of the plasmonic interferometer; and a sample
holder for disposing a sample to be analyzed onto the first surface
of the plasmonic interferometer; wherein the sample comprises an
oxidative or chromogenic dye selected to absorb particular light
frequencies.
Description
BACKGROUND
The invention is generally directed to systems and methods for
high-throughput, real-time detection of analytes in fluids, for
example, bodily fluids (e.g. cytokines in blood, glucose in saliva,
tears and blood, etc.).
Current detection and screening techniques use low throughput,
highly selective, non-scalable methods, which require labeling of
the target molecule with a fluorophore to tag the specific molecule
under study. These methods have several drawbacks: (1) prior
knowledge of the molecule to be sensed is necessary, (2)
modification of its structure is often needed to incorporate the
tag, and (3) the tag molecule can change the way the primary
molecule binds to other molecules, reducing the accuracy of an
assay. Under alternative aspects of the present disclosure,
label-free sensing is achieved using surface plasmon resonance
(SPR) in thin metal films functionalized with specific linkers to
selectively capture the analyte to be detected.
Typical SPR implementations rely on a prism or metallic grating
(such as groove, slit and hole arrays) to couple the incident beam
into propagating surface plasmon polaritons (SPPs), using light
incident at a wavelength-specific angle. Also widely used are
localized surface plasmon resonances (L-SPRs) in metal
nanoparticles producing resonant scattering and extinction at
specific frequencies. Given the resonant nature of the SPP
excitation, practical implementations of SPR-based sensing schemes
are limited in the number and range of wavelengths that can be used
to sense the presence of analytes, thus further limiting their
spectroscopic capabilities. Furthermore, SPR-based systems require
large-volume samples and are limited to the detection of chemicals
one at a time.
The dispersion relation of SPP waves shows longer wave vectors
compared to electromagnetic waves traveling in dielectric materials
(See FIG. 1a). This is why light incident upon a flat metal surface
cannot excite surface plasmon polaritons directly (See FIG.
1b).
According to aspects of the present disclosure, excitation of SPP
waves can be achieved by nano-corrugations, patterned on a flat
metal surface (See FIGS. 1c-e), for example, grooves, slits, holes,
or any surface plasmon launcher, which is any structure that can
act as a SPP localized source.
FIG. 2a shows a commercially available Surface Plasmon Resonance
(SPR) biochip. By measuring the angular shift of this
characteristic band, detection of a refractive index change is
accomplished. This approach leads to table-top instruments, where a
large sensing area is needed to detect only one specific molecule
per measurement session, thus limiting the throughput. Moreover,
SPR sensors are monochromatic, in that they typically employ a
single illumination wavelength, and they cannot determine the
spectral fingerprints of the analyte.
Alternative approaches to label-free detection include
fiber-optics, dielectric waveguides, nanowires, biochips,
mechanical cantilevers, microring resonators, but none of the
previous methods has shown significant throughput capabilities. For
example, FIG. 2b shows an ultra high-Q microtoroid sensor.
Label-free detection can also be realized by measuring some optical
properties of functionalized noble-metal nanoparticles, such as
Surface Enhanced Raman Scattering (SERS), or Mie scattering and
light extinction. For example, FIG. 2c shows a nanoscale biosensor.
Unfortunately, the detection throughput is strongly limited by the
difficulty to reliably address the response of several
nanoparticles at once, by the challenging task to achieve uniform
coating of all nanoparticles with a linker, and reproducible sensor
response.
FIG. 2d shows a typical implementation of SPR, which involves a
prism to excite the surface plasmons at the functionalized
metal/dielectric interface, which happens when the incident angle
is precisely chosen at a specified wavelength. In order to improve
throughput, other techniques, such as SPR imaging, have been
developed that allow several device cells (.about.10.sup.2) to be
used in parallel to image the binding interaction and monitor
intensity variations caused by a refractive index change in each
cell.
Another method to excite SPR employs metallic gratings (such as
hole, slit and groove arrays) as shown in FIGS. 2e-g. This method
is based on the idea that the prism is replaced by a periodic array
of scatterers etched in a metal film. Excitation of surface
plasmons occurs at those resonant wavelengths (or angles, given a
specific incident wavelength) satisfying the grating coupling
condition. Therefore, reflection and transmission spectra from
these devices are also characterized by sharp peaks corresponding
to the wavelength- or angle-specific resonant condition. Metal
nanoparticles are also widely used, which are characterized by
sharp spectroscopic features in their scattering and extinction
spectra, so-called localized surface plasmon resonances (LSPRs).
Upon binding of the analyte of interest to the functionalized
nanoparticle surface, a shift in the peak position of the LSPR is
observed and the relative wavelength shift can be used to quantify
the amount of analyte adsorbed at the surface. Given the "resonant"
nature of the surface plasmon excitation, all the previous
approaches involving SPR (prism-, grating-coupled, or localized)
are limited in the number of wavelengths that can be used to sense
the presence of the analyte. Therefore, the refractive index
associated to the analyte can be measured only at one specific
wavelength, thus strongly limiting the spectroscopic capabilities
of any currently available SPR technique.
According to aspects of the present disclosure, a nanoscale
plasmonic interferometer in one manifestation comprises of two
grooves flanking a slit in a silver film is provided (see FIG.
2g).
A plasmonic-based device that accurately measures chemical and
biological analytes in real-time is provided. Chemical analytes
include but are not limited to dielectric materials such as
semiconductor quantum dots (QDs) (see FIG. 3a), analytes embedded
in thin films, or molecules in a gas or liquid phase. Biological
analytes include but are not limited to proteins (e.g. cytokines in
blood serum) and small molecules (e.g. glucose in bodily fluids)
(see FIG. 3b-d).
In addition to a better understanding of light-matter interaction
at the nanoscale, the disclosed methods and systems impact the
throughput capabilities of several analyses and assays relevant to
human health and currently used in the life sciences, and serve as
an alternative high-throughput scheme for faster drug discovery, as
well as more efficient identification and screening of novel
therapies.
SUMMARY OF THE INVENTION
According to aspects of the present disclosure, a system for
detecting an analyte is provided. The system includes a plasmonic
interferometer comprising a first surface having a first scattering
element and a second scattering element and a aperture disposed
between the first scattering element and the second scattering
element to define a first distance between the aperture and the
first scattering element and a second distance between the aperture
and the second scattering element, wherein the first and second
distances are selected to provide controlled interference of light
at the aperture. The system also includes a light source for
illuminating the first surface of the plasmonic interferometer, a
detector positioned for detecting light transmitted through the
aperture, and a sample holder for disposing a sample to be analyzed
onto the first surface of the plasmonic interferometer.
According to alternative aspects of the present disclosure, a
method of real-time detection of an analyte is provided. The method
includes the steps of providing a plasmonic interferometer,
applying a sample to be analyzed on the first surface of the
plasmonic interferometer, illuminating the plasmonic interferometer
with the light source, measuring a light property of a composite
light transmitted through an aperture, and determining a
characteristic of an analyte of interest based on the measured
light intensity.
According to alternative aspects of the present disclosure, a
system for detecting a plurality of analytes includes a plurality
of plasmonic interferometers. Each plasmonic interferometer
includes a first surface having a first scattering element and a
second scattering element and an aperture disposed between the
first scattering element and the second scattering element to
define a first distance between the aperture and the first
scattering element and a second distance between the aperture and
the second scattering element. The first and second distances are
selected to provide constructive interference of light at the
aperture. The system can also include a light source for
illuminating the first surface of each of the plurality of
plasmonic interferometers, a plurality of detectors positioned for
detecting light transmitted through the aperture of each plasmonic
interferometer, and a plurality of sample holders, each sample
holder disposing a sample to be analyzed onto the first surface of
each plasmonic interferometer.
According to alternative aspects of the present disclosure, a
system for detecting an analyte includes a plasmonic
interferometer, which includes a first surface having a first
scattering element and a second scattering element and an aperture
disposed between the first scattering element and the second
scattering element to define a first distance between the aperture
and the first scattering element and a second distance between the
aperture and the second scattering element, wherein the first and
second distances are selected to provide controlled interference of
light at the aperture. The system can also include a light source
for illuminating the first surface of the plasmonic interferometer,
a detector positioned for detecting light transmitted through the
aperture; and a sample holder for disposing a sample to be analyzed
onto the first surface of the plasmonic interferometer. The sample
can comprise an oxidative or chromogenic dye selected to absorb
particular light frequencies.
According to alternative aspects of the present disclosure, a
method of real-time detection of an analyte includes the steps of
providing a plasmonic interferometer and applying a sample to be
analyzed on the first surface of the plasmonic interferometer,
wherein the sample comprises an oxidative or chromogenic dye
selected to absorb particular light frequencies. The method also
includes the steps of illuminating the plasmonic interferometer
with the light source, measuring a light property of a composite
light transmitted through an aperture, and determining a
characteristic of an analyte of interest based on the measured
light property.
According to alternative aspects of the present disclosure, a
method of spectroscopic measurements of the dispersion relation and
optical constants of dielectric materials in gaseous, liquid, or
solid form includes the steps of providing a plasmonic
interferometer, applying a sample to be analyzed on the first
surface of the plasmonic interferometer, wherein the sample
comprises at least one of a dielectric materials or a mixture
thereof of dielectric material, illuminating the plasmonic
interferometer with a light source, measuring a light property of a
composite light transmitted through the slit aperture, and
determining a characteristic of an analyte of interest in the
sample based on the measured light property.
According to aspects of the present disclosure, methods of
fabrication of microarrays of plasmonic interferometers are
provided, by designing, modeling, fabricating and characterizing
metal films patterned according to various periodic, random and
quasi-periodic nano-hole, and slit and groove arrays.
According to aspects of the present disclosure, microarrays of
plasmonic interferometers are provided, for example, by patterning
metal films according to various periodic, random and
quasi-periodic nano-hole, and slit and groove arrays.
According to alternative aspects of the present disclosure, methods
of development and implementation of surface chemistry by which to
attach selective sensing elements for targeted cytokines and
glucose are provided.
According to alternative aspects of the present disclosure, methods
of device fabrication and optimization for effective cytokine
detection in blood serum and glucose detection in saliva are
provided.
According to alternative aspects of the present disclosure, devices
for effective cytokine detection in blood serum and glucose
detection in saliva are provided.
According to alternative aspects of the present disclosure, methods
for extraction of fingerprint (dispersion relation) and composition
of the analyte are provided.
DESCRIPTION OF THE FIGURES
The following figures are presented for the purpose of illustration
only, and are not intended to be limiting:
FIG. 1a is a plot of energy (eV) v. k.sub.x (nm.sup.-1) showing the
surface plasmon dispersion relation for Ag/SiO.sub.2, (b) is an
illustration of light incident upon a flat metal surface.
FIGS. 1c-e show nanoscale corrugations (e.g. groove, slit and hole)
patterned on a flat metal surface that can excite surface plasmon
polaritons in counter-propagating directions.
FIGS. 2a-c show a prior art surface plasmon resonance biochip, a
microring resonator, and a metal nanoparticle coated with specific
linkers, respectively.
FIGS. 2d-g show a prism coupling, grating coupling, groove-slit and
groove-slit-groove plasmonic interferometer sensing scheme,
respectively, using surface linkers capable of specific
binding.
FIG. 3 shows schematics of the general applications of the
plasmonic interferometer including: (a) an in-situ spectroscopic
ellipsometer for measuring the dispersion relation and optical
constants of dielectric materials at the metal surface, (b) a
plasmonic interferometer for small molecule detection (e.g.
glucose, drugs) (c) a plasmonic interferometer coupled with
oxidative and chromogenic dyes for enhanced sensing, and (d) a
plasmonic interferometer for protein (e.g. cytokines) sensing.
FIGS. 4a-c show SEM micrographs of plasmonic interferometers
according to aspects of the present disclosure, in which the
plasmonic interferometer includes (a) a single groove/slit
arrangement, (b) a two grooves and a slit (GSG) having different
groove/slit spacings, p.sub.1 and p.sub.2, respectively, and (c) a
two grooves and a slit (GSG) having different groove/slit spacings,
p.sub.1 and p.sub.2, respectively, (d) is a schematic illustration
of a GSG plasmonic interferometer illustrating groove slit
distances p.sub.1 and p.sub.2, (e) shows the light intensity
spectra transmitted through an isolated slit and through the slit
of a GSG interferometer, and (f) shows the experimental and
simulated normalized per-slit transmission spectra.
FIG. 5 shows normalized per-slit transmitted intensity spectra
(experimental and simulated) for two GSG plasmonic interferometers,
according to aspects of the present disclosure.
FIG. 6 shows predicted normalized light transmission, as a function
of incident wavelength, through a slit aperture in a plasmonic
interferometer immersed in water, according to aspects of the
present disclosure.
FIG. 7 shows predicted relative percent change in light intensity
transmitted through the slit of two plasmonic GSG interferometers,
as a function of refractive index change induced by the presence of
cytokines.
FIG. 8 shows a simulated 2D color map of normalized light intensity
transmitted through the slit of several plasmonic interferometers
with p.sub.1=0.57 .mu.m, as a function of groove-slit arm length
p.sub.2 (horizontal axis) and wavelength .lamda. (vertical axis).
Typical normalized transmitted intensity plots obtained by
horizontal or vertical cuts across the color map (indicated by gray
boxes) are also shown.
FIG. 9a-l shows color maps of experimental and simulated normalized
light transmission spectra collected through plasmonic
interferometers at different combinations of p.sub.1 and p.sub.2
according to aspects of the present disclosure.
FIG. 10 shows schematics of (a) uncoated and (b) CdSe coated
plasmonic interferometers, (c-d) charts of measured transmitted
intensities with and without coating at 687.9 nm and 514.5 nm,
respectively, and (e) a schematic of a CdSe quantum dot used to
coat the plasmonic interferometer surface.
FIG. 11 shows (a) the effective SPP excitation efficiency in Ag/air
and Ag/water interfaces as a function of wavelength; (b) the
extracted dielectric constant of Ag and water as a function of
wavelength.
FIG. 12 shows (a) normalized per-slit transmitted intensity spectra
of a GSG device measured at various concentrations of glucose in
water; and (b) relative intensity change spectra normalized to pure
water at various concentrations of glucose in water.
FIG. 13a-b shows calibration curves for a plasmonic
interferometers.
FIG. 14a-d show wavelength shift and relative intensity change
versus glucose concentration, for four plasmonic interferometers,
respectively.
FIG. 15 shows the selective reaction scheme for glucose detection
that utilizes the coupling of a chromogenic dye and plasmonic
interferometry for enhanced selectivity and specificity.
FIG. 16 shows (a) relative intensity changes based on a dye-coupled
exemplary glucose detection method, according to aspects of the
present disclosure; and (b) the calibration curve for this method
comparing with the performance of a device not using the dye
method.
FIG. 17 shows a schematic for immobilization of a selective protein
to a targeted analyte (e.g. human anti-IL6 (hAnti-IL6 for cytokine
detection, glucose oxidase (GOx) for glucose detection, etc.) onto
a metal substrate that supports surface plasmon generation (e.g.
Ag, Au, etc.) using EDC/NHS coupling.
FIG. 18a shows the temporal evolution of human anti-IL6 antibody
(hAnti-IL6) immobilized onto gold-coated quartz substrate via
EDC/NHS coupling chemistry (11-MUA=thiol coupling agent,
EDC/NHS=free coupling agent, Anti-ILS=coupled human anti-IL6
antibody); and FIG. 18b shows temporal evolution of glucose oxidase
(GOx) monolayer immobilized onto a silver-coated quartz substrate
via EDC/NHS coupling chemistry.
FIG. 19 shows (a) the contact angles for the silver film with and
without Al.sub.2O.sub.3 protection; and (b) the normalized
transmitted intensity in water for a device protected by the
Al.sub.2O.sub.3 film before and after the dye exposure, according
to aspects of the present disclosure.
FIG. 20 shows an exemplary arrangement of plasmonic interferometers
in an array, according to aspects of the present disclosure.
FIGS. 21a-b show the skin depth and propagation length of surface
plasmons, respectively, according to aspects of the present
disclosure.
FIG. 22 shows a three-dimensional computer-aided design and
rendering of a chip with plasmonic interferometers used in
detecting multiple analytes, according to aspects of the present
disclosure.
FIGS. 23a-b show a schematic and a photograph of a prototype
plasmonic interferometer, respectively, according to aspects of the
present disclosure.
FIG. 24 1-8 illustrates step-by-step instructions to use a
self-monitoring glucose meter, according to aspects of the present
disclosure.
DETAILED DESCRIPTION OF THE INVENTION
Optical interferometry is a valuable tool used in a variety of
areas ranging from astronomy, to fiber optics and biomedical
imaging. Typical implementations of optical interferometers employ
cavities with dimensions ranging from hundreds of micrometers to
meters, such as those used in lasers and optical coherence
tomography, and as long as several kilometers, such as those used
in the laser interferometer observatory where the existence of
gravitational waves (LIGO) has been revealed.
Plasmonics is a rapidly emerging field of nanophotonics enabling
the guiding and manipulation of light in devices with a footprint
much smaller than the wavelength of the incident light. Plasmonics
focuses on the ability of metal nanostructures to manipulate light
at the nanoscale. For example, by using nanocorrugations etched in
a metal film, light at optical frequencies can be coupled to
surface plasmon polaritons (SPPs). SPPs are electromagnetic waves
coupled to oscillations of free electrons in a metal. SPPs are
guided electromagnetic waves that propagate along a metal
dielectric interface with high field confinements at the interface.
SPPs are confined at the metal surface and are very sensitive to
small changes in the refractive index of the dielectric (e.g.,
aqueous solutions with analytes).
SPPs are characterized both by maximum field amplitudes at the
metal surface and by wavelengths much shorter than the incident
wavelength. SPPs have the potential therefore to encode a huge
amount of information, indeed larger than in devices with
dimensions comparable to or even smaller than modern
microelectronic transistors. Moreover, being confined at the metal
surface, SPPs are very sensitive to the dielectric properties of
the materials on which they propagate. This property allows for
increased interaction with light emitters, enhanced field
intensities at the surface of metal nanoparticles, extraordinary
transmission through periodic hole arrays, higher absorption
efficiencies in ultra-thin dielectric layers, as well as
superlensing and negative refraction for cloaking applications at
visible wavelengths. Moreover, SPPs can be used to sense the
presence of chemical and biological analytes. According to aspects
of the present disclosure, plasmonic sensors employing
interferometry at the micro- and nano-scale are provided for
sensing the presence of chemical and biological analytes.
According to aspects of the present disclosure, optical
interferometry applications scaled to the nano- and micro-meter
scale are provided, using novel plasmonic interferometers as the
transducers to detect and convert the interaction of an analyte
(e.g., cytokines in blood serum, glucose in bodily fluids, etc.)
with the sensor surface into a measurable variation of light
intensity.
According to aspects of the present disclose, the proposed
interferometric approach benefits from the use of surface plasmons
that are excited by diffractive scattering of incident light by
subwavelength grooves. The proposed sensing scheme can retain the
attractiveness of real-time and label-free sensing of conventional
SPR.
The nanoscale groove can be used to excite surface plasmons in a
broad wavelength range, keeping the angle of incidence constant
(for example normal incidence), which constitutes a substantial
advantage compared to other grating- and prism-coupling strategies
where precise determination and setting of the coupling angle is
required for every trial. The proposed method can enable the
determination of the refractive index for various analytes as a
function of wavelength. Sensitivity of the sensor can be enhanced
by simply tuning the incident wavelength or varying the
interferometer arm lengths.
According to aspects of the present disclosure, a device based on
novel plasmonic architecture can accurately and rapidly measure the
level of analytes in a sample in real time. As is demonstrated
herein, analytes can be detected at extremely low levels, e.g., at
nano-gram, pico-gram and femto-gram levels.
Furthermore, the fundamental science and technology of the
disclosed invention can transform to all areas where real-time,
sensitive detection of dilute analytes are needed.
The proposed design for plasmonic interferometry is fabricated at
the nano scale, employing periodic and quasi-periodic arrays of
sub-micrometer scale holes, slits, and grooves (both linear and
curvilinear), and can be coupled to surface chemistries that are
specific for the analyte of interest. Under aspects of the specific
disclosure, a dense microarray of plasmonic interferometers can
provide high-throughput, real-time data on analyte levels. In
certain aspects, detectors based on plasmonic interferometers can
provide high-throughput, real-time data on diluted analytes at low
concentrations, such as cytokines in blood serum, glucose in bodily
fluids, etc., capabilities not possible with current
technology.
According to aspects of the present disclosure, methods of
fabrication of microarrays of plasmonic interferometers are
provided, by designing, modeling, fabricating and characterizing
metal films patterned according to various periodic, random and
quasi-periodic nano-hole, slit and groove arrays.
According to aspects of the present disclosure, microarrays of
plasmonic interferometers are provided, for example by patterning
metal films according to various periodic, random and
quasi-periodic nano-hole, and slit and groove arrays.
Detection of extremely dilute chemical and biological species is
generally accomplished using low throughput, non-scalable methods
that often rely on target labeling to reveal the presence of
specific molecules within a single device. Under aspects of the
present disclosure, systems and methods are provided for chemical
and biological sensing that consists of millions of nano- and
micron-scale optical interferometers per centimeter squared, each
working as an individually addressable sensor, integrated on a
single device.
The detector element is a plasmonic interferometer able to transmit
light through a nano-aperture and acting as the transducer. Any
variation in analyte levels will determine a change in refractive
index and as a consequence the light intensity will vary. Light
intensity transmitted through the interferometer will be measured
as a function of incident wavelength to determine the spectroscopic
fingerprints of the analyte. Moreover, the geometrical parameters
can be varied to generate a dense array of such interferometers,
working in parallel and individually addressable for
high-throughput screening. Scaling the device footprint can be
possible using a CCD camera and optical elements. A microprocessor
interfacing with the device can perform real-time analysis of the
data and display the results.
The microarrays of plasmonic interferometers are prepared using
conductive surfaces, such as gold, silver, copper, aluminum, and
indium tin oxide and the like. The surfaces can be modified to
provide interaction and binding of the analyte of interest using
well established methods. Surface chemistries include metal-thiol
self-assembled monolayers and subsequent chemical reactions at
their end-groups as well as electrodeposited polymers and their
subsequent surface modification. Surfaces modified by these
chemistries can be used for electrochemical sensing (e.g., vitamin
D and glucose detection), bioelectrocatalysis (e.g., oxygen
reduction), and cellular communication (e.g., electrically
stimulated nerve cell adhesion and neurite extension).
According to aspects of the present disclosure, nanoscale plasmonic
interferometers comprising two grooves flanking a slit in a silver
film are provided. The two grooves can scatter a normally incident
white light beam into multifrequency SPPs, counter-propagating at
the metal/dielectric interface. The field amplitudes of the two SPP
waves interfere with the incident field at the slit location, thus
causing a modulation in the light intensity transmitted through the
slit. The transmitted intensity depends on wavelength, refractive
index, and it can also be tuned by simply varying the groove-slit
separation distances (i.e., the lengths of the two interferometer
arms). Since SPPs are strongly confined at the metal/dielectric
interface, a far-field measurement of the light intensity
transmitted through the slit carries information about the
near-field interaction of the SPPs with the dielectric material,
useful to determine the refractive index of unknown chemical
analytes and their concentration in solution, only a few tens or
hundreds of nanometers above the metal surface. Furthermore, SPPs
can be generated at multiple frequencies simultaneously using the
same groove and incoupling angle, thus enabling operation of the
plasmonic interferometer event at frequencies far from the metal
resonance, and extraction of the analyte dispersion relation in a
broad wavelength range. Hence, plasmonic interferometers offer a
novel scheme for SPP excitation and refractive index detection
using SPP interference, retaining real-time and label-free sensing
capabilities of existing technologies.
An exemplary plasmonic interferometer is shown in FIG. 2g. The
device includes two grooves located on opposite sides of an
aperture, shown here as a slit. The first and second grooves are
spaced apart from the slit by distances p.sub.1 and p.sub.2,
respectively. FIG. 2g schematically shows that the plasmonic
interferometer works by optical interference between the two
counter-propagating SPP waves (E.sub.spp1, E.sub.ssp2) and the
incident beam at the slit location (E.sub.0). Light incident upon
the left-side groove generates SPPs propagating toward the slit. At
the slit location, and for each frequency, the SPP (with amplitude
E.sub.SPP1) will interfere with the coherent incident beam
(E.sub.0). Light incident on the right groove also excites an SPP
with amplitude E.sub.SPP2, traveling along the metal surface and
interfering with the incident beam and the other SPP wave at the
slit location. The light that is transmitted through the slit is
characteristic of the SPP waves and the dielectric medium through
which the SPP waves propagate. FIG. 2f is a schematic illustration
of the device (only a single groove is shown for simplicity), in
which the surface has been modified to provide a surface chemistry
(indicated by tethered `horseshoe` 100) capable of binding the
target analyte (indicated as `starburst` 110).
Fabrication and Modeling the Optical Response of the Plasmonic
Interferometers.
According to alternative aspects of the present disclosure,
plasmonic interferometers were fabricated by focused ion beam
milling on a 300 nm-thick Ag film, previously evaporated on a clean
glass slide. Different kinds of plasmonic interferometers were
designed and fabricated, consisting, for example, of a groove-slit
pair with separation distance p, and a novel groove-slit-groove
design, with slit-groove separation distances (i.e. interferometer
arms) p1 and p2, varying in the range 250 nm-2 .mu.m, in steps of
25 nm. Moreover, several individual slits were etched on the same
film to serve as a reference. FIGS. 4a-c show SEM micrographs of
representative devices. The grooves and slits were 200 nm and 100
nm wide, 50 nm and 300 nm deep, respectively, and 10 .mu.m long. In
FIG. 4a, the separation distance between the groove and the slit is
p=250 nm, while in FIG. 4b, the slit-groove separation distances
are p.sub.1=1670 nm and p.sub.2=250 nm. FIG. 4c shows another
groove-slit-groove interferometers with different slit-groove
separation distances. The dimensions of the slit and groove affect
the performance and sensitivity of the interferometer. The
dimensions can vary, for example, to excite particular wavelengths
or to adjust the launching phase of the surface plasmons. The
length of the slit and grooves can vary. The length can range, for
example, between 500 nm and 100 .mu.m. The width of the slit and
grooves can also vary. The width can vary between 50 nm and 500 nm.
The depth of the grooves can also vary. The depth can range, for
example, between 5 nm and 300 nm.
FIG. 4d shows another schematic an interferometer according to
aspects of the present disclosure. Light incident upon the
left-side groove generates a collective oscillation of the
conduction electrons in the metal film that can propagate toward
the slit, in the form of a surface wave, along the metal/dielectric
interface. This SPP wave has a complex amplitude (E.sub.SPP1),
whose phase and amplitude can be affected by any chemical analyte
encountered along the optical path. Light incident on the right
groove goes through the same process and has an amplitude,
E.sub.SSP2. Useful information about the kind and quantity of
analytes adsorbed on the surface can be retrieved by interfering
the surface wave with the incident beam (E.sub.0) at the slit
location, which causes a change in the total light field
transmitted at the output mouth of the slit (E.sub.T). As a result
of the interference process between the surface plasmon and the
incident light beam, the light intensity transmitted through the
slit can be modulated, i.e. either enhanced or suppressed,
depending on whether a constructive or destructive interference
occurs. By measuring the light intensity transmitted through the
slit as a function of wavelength and monitoring the change thereof
caused by the presence of an analyte, we can estimate the amount
and fingerprints of the adsorbed chemical species.
FIG. 4e shows the representative spectra for light intensity
transmitted through an isolated nano-slit and through the nano-slit
of a groove-slit-groove interferometer, with arms p.sub.1=0.57
.mu.m and p.sub.2=1.85 .mu.m, respectively. A halogen light source
was aligned to the optical axis of a Nikon Ti Eclipse inverted
microscope. The beam was collimated at normal incident onto the
sample surface with respect to the silver/air interface. The light
intensity transmitted through the slit of the device was collected
by an objective lens and sent to a single grating monochromator,
then detected with a digital CCD array. In order to detect the
modulation effects caused by the interference between the surface
plasmons and the incident beam, the transmission spectrum was
normalized by dividing the light intensity transmitted through the
groove-slit-groove interferometer by the transmission spectrum of
an isolated slit. The resulting normalized per-slit transmitted
intensity spectrum is reported in FIG. 4f. Compared to an isolated
slit, light intensity can be enhanced or suppressed because of
constructive or destructive interference. The observed intensity
modulation in the normalized transmission spectrum for a GSG device
results from interference (at the slit location) between the two
counter-propagating SPPs originating from in-plane diffractive
scattering of light at the two grooves, and the incident beam. The
slit in between the two grooves effectively acts as a "spatial
mixer" of the three field amplitudes (incidence beam plus the two
SPP waves generated by diffractive scattering). The light intensity
transmitted through the slit contains information of the relative
phase difference and amplitude of the different beams. FIG. 4f also
reports the simulated normalized per-slit transmission through the
slit of the GSG plasmonic interferometer (dashed line) using the
model developed above. The agreement between the experimental and
simulated normalized per-slit transmission spectra, demonstrates
that transmission maxima and minima result from constructive and
destructive interference between the incident beam and the two
counter-propagating SPPs excited by diffractive scattering at each
groove position. SPPs can therefore be generated at multiple
wavelengths using the same incident angle, a novel capability not
possible in SPR techniques based on prism- and
grating-coupling.
A total of 568 devices over an area of 3.5.times.4.0 mm.sup.2 were
etched using an automated scripting routine implemented to control
the focused ion beam parameters and stage position during milling.
This corresponds to a density of >4,000 devices per centimeter
squared. The device density range can be as low as one single
interferometer per chip, or as high as one million (or greater) per
millimeter squared. After fabrication, light transmitted intensity
through each device was measured using broadband illumination at
normal incidence, and a monochromator to disperse the transmitted
intensity onto a CCD camera to detect a real-time transmission
spectrum.
FIG. 5 shows the experimental (solid lines) and simulated (dashed
lines) normalized transmitted intensity spectra for two plasmonic
interferometers with constant p.sub.1=0.57 .mu.m and different
p.sub.2 lengths: 1.85 .mu.m (black lines) and 5.70 .mu.m (red
lines). From this figure, a greater number of intensity maxima and
minima can be observed by increasing p.sub.2, resulting in more
wavelengths at which constructive and destructive interference
occurs. These results suggest that the device sensitivity can be
improved at various wavelengths by simply increasing the
groove-slit separation distance. This distance is only limited by
the ohmic and scattering losses of SPPs, which in general reduce
the SPP propagation length, and by the spatial and temporal degree
of coherence between the SPPs and the incident beam, needed to
determine the interference effect.
An SPP interference model was developed to predict the optical
response for the plasmonic interferometer, extending reported
models dealing with groove diffraction and SPP interference. As
shown in FIG. 4d, light incident upon the left-side groove
generates SPPs propagating toward the slit. At the slit location,
and for each frequency, the SPP (with complex amplitude E.sub.SPP1)
will interfere with the coherent incident beam (E.sub.0). Light
incident on the right groove also excites an SPP with amplitude
E.sub.SPP2, traveling along the metal surface and interfering with
the incident beam and the other SPP wave at the slit location.
Though the slit can also generate SPPs, these are mainly scattered
back in free space by out-of-plane scattering once they reach the
neighboring grooves. Therefore, slit-generated SPPs do not
contribute significantly to the transmitted intensity through the
same slit. Accordingly, for this model, only the SPPs originating
from the two grooves will be considered. This assumption is
verified by the excellent agreement between simulated and
experimental results.
The resulting total transmitted intensity through the slit of a GSG
two-arm plasmonic interferometer is given by
I.sub.T=|E.sub.T|.sup.2=I.sub.S|1+.beta..sub.1e.sup.i.phi..sup.1+.beta..s-
ub.2e.sup.i.phi..sup.2|.sup.2
where I.sub.S is the light intensity transmitted through an
isolated slit with identical width and length, subscripts 1 and 2
denote the SPP contributions originating from the left and right
groove, respectively, .beta..sub.1,2 accounts for the effective
efficiency of SPP excitation via diffractive scattering by each
groove, and .phi..sub.1,2 is the total phase shift of the SPP
including a complex phase accounting for propagation and absorption
in the metal and dielectric material, and a scattering phase
accrued by the SPP upon excitation by each groove, given by
.phi..times..pi..lamda..times..function..-+..times..times..times..theta..-
phi..times..times. ##EQU00001##
where .lamda. is the free-space wavelength of the incident beam,
p.sub.1,2 is the groove-slit distance, .theta. is the angle between
the incident light beam and the normal to the sample surface
(.theta.=0 herein), n.sub.SPP is the complex refractive index of
the SPP given by n.sub.SPP=[.di-elect cons..sub.m.di-elect
cons..sub.d/(.di-elect cons..sub.m+.di-elect cons..sub.d)] where
.di-elect cons..sub.m is the complex dielectric constant of the
metal, .di-elect cons..sub.d is the complex dielectric constant of
the material above the metal, n.sub.d is the refractive index of
the dielectric material, and .phi..sub.G1,2 is an additional phase
shift due to the initial scattering by the groove. The SPP
propagative phase is affected by several tunable parameters, such
as the incident wavelength (.lamda.), the distance between the slit
and the grooves (p.sub.1,2), and the refractive index of the
dielectric material, n.sub.d=.di-elect cons..sub.d.sup.1/2.
If the two grooves are identical and the dielectric material on top
of each is the same, then the SPP excitation efficiencies and
scattering phases at each groove are the same, i.e.,
.beta..sub.1=.beta..sub.2=.beta. and
.phi..sub.G1=.phi..sub.G2=.phi..sub.G. The transmitted intensity
through the slit (normalized to the light intensity transmitted
through an isolated slit) then becomes
I.sub.T/I.sub.S=|1+.beta.{e.sup.i[p.sup.1.sup.(k.sup.SPP.sup.-k sin
.theta.)+.phi..sup.G.sup.]+e.sup.i[p.sup.2.sup.(k.sup.SPP.sup.+k
sin .theta.)+.phi..sup.G.sup.]}|.sup.2
where k.sub.SPP=2.pi.n.sub.SPP/.lamda. and k=2.pi.n.sub.d/.lamda..
As a result of the interference process between the SPP waves and
the incident beam, the light intensity transmitted through the slit
can be either enhanced or suppressed, depending on whether
constructive or destructive interference occurs. In comparison,
light transmitted through the slit of a plasmonic interferometer
consisting of only one groove-slit pair will be the result of a
two-beam interference (SPPs from the two grooves plus the incident
beam), which would result in reduced beatings and
constructive/destructive interference effects. The GSG plasmonic
interferometers clearly show better sensitivity compared to GS
devices and were chosen accordingly for the sensing experiments. To
verify the model, the plasmonic interferometers were illuminated
using a collimated, broadband light source normally incident upon
the sample surface, with a power density of .about.10.sup.-2
W/cm.sup.2. A 40.times. objective lens was used to collect the
far-field light intensity transmitted through the slit of each
plasmonic interferometer, then dispersed using a single-grating
spectrograph and detected by a CCD camera. Spectral resolution of
the optical setup was .about.0.4 nm; the number of counts and
acquired spectra per experiment were adjusted to ensure a
statistical error of <0.1% in the measured transmitted
intensity.
FIG. 6 shows a proposed extension of the model to calculate the
normalized transmitted intensity for a groove-slit-groove
interferometer immersed in water. Specifically, FIG. 6 shows the
predicted normalized light transmission through a slit aperture in
a groove-slit-groove interferometer, with p.sub.1=800 nm and
p.sub.2=816 nm, as a function of incident wavelength. The black
curve has been calculated by using the dispersive dielectric
constant of de-ionized water and the optical constants of Ag. Upon
introduction of small quantities of analyte in water corresponding
to a refractive index change .DELTA.n=0.2, a clear modification of
the transmission curve occurs. In particular, it is possible to
notice that the light intensity at 532 nm goes from a minimum to a
maximum upon introduction of the analyte in water. The relative
change of intensity before and after index change is .about.40.
FIG. 7 shows the predicted relative percent change in light
intensity transmitted through the slit of two plasmonic
groove-slit-groove interferometers, as a function of refractive
index change induced by the presence of cytokines Specifically, the
gray line in FIG. 7 reports a more systematic study of the percent
change in transmitted light intensity, defined as
[I.sub.T(.DELTA.n)-I.sub.T(0)]/I.sub.T(0), recorded at 532 nm for
the same device as a function of refractive index change. It is
worth noting that in going from .DELTA.n=0.002 to .DELTA.n=0.2, the
interferometer response varies from 1% to 4,000% relative change in
light transmitted intensity, which corresponds to a relative gain
of 2.times.10.sup.4 per refractive index unit. The sensitivity can
be further enhanced by using longer wavelengths and longer
slit-groove separation distances. The square-identified line in
FIG. 7 reports the relative change in light transmitted intensity
for a device with p.sub.1=21.6 .mu.m and p.sub.2=27.5 .mu.m at a
wavelength .lamda.=1000 nm.
For a refractive index change of 2.times.10.sup.-3 the
interferometer with longer arms has a 40 times higher signal
response, with a gain of 7.times.10.sup.4. Further analysis of the
interferograms generated by a properly functionalized optical
biosensor can determine the presence of cytokines and can generate
a calibration curve (similar to the one reported in FIG. 7) as a
function of cytokine concentration (in the range of 1-100 nM,
corresponding to an estimated refractive index change of
.DELTA.n=3.times.10.sup.-4-3.times.10.sup.-2) with extremely high
sensitivity and in real time.
FIG. 8 shows a simulated color map of normalized per-slit light
intensity transmitted through the slit of a series of plasmonic
interferometers in air, at a given p.sub.1=0.57 .mu.m and varying
p.sub.2=0.25-2.0 .mu.m, as a function of wavelength (400-800 nm).
To construct this color map, normalized transmission spectra or
"wavelength profiles" (vertical gray box in FIG. 8) for plasmonic
interferometers with varying p.sub.1 and p.sub.2 were stacked
according to increasing p.sub.2. The color of each pixel in the
maps represents the measured normalized transmitted intensity
I.sub.T for a specific combination of slit-groove separation
distances and wavelength, i.e. we are plotting I.sub.T (p.sub.1,
p.sub.2, .lamda.). The color bar shows that light transmission
through a slit flanked by the two grooves can be enhanced by as
much as a factor 2.5 compared to an isolated slit, and suppressed
by the same amount. From this map, a horizontal "cut" (for example
at 600 nm, as indicated by the horizontal gray box in FIG. 8) can
reveal an intensity profile as a function of arm length p.sub.2,
for a given incident wavelength. According to equations mentioned
above, the difference between intensity maxima and minima can be
shown to be proportional to the SPP excitation efficiency from the
groove. Therefore, such horizontal cuts in the experimental color
maps are useful in determining the SPP excitation efficiency at
various wavelengths.
FIG. 9 shows a comparison between experimental results and
simulation results for normalized light transmission spectra
through the slit of hundreds of plasmonic interferometers
consisting of a slit aperture flanked by two grooves located at
different slit-groove separation distances. Each of the six panels
reports color maps of transmission spectra through devices with
varying slit-groove separation distances (p.sub.1, p.sub.2)
normalized by the spectrum of an isolated reference slit. In order
to understand the interference phenomenon, we have to realize that
each groove acts as a localized, efficient source of surface
plasmon polaritons (SPPs) traveling toward the neighboring slit. At
the slit location, the two propagating SPPs interfere with the
incident light beam at the slit location, thus causing either
constructive or destructive interference. Under aspects of the
present disclosure, an analytical model can describe the
interference process. The resulting color maps are presented in the
bottom panel of FIG. 9. These color maps show remarkably good
agreement with the experimentally determined data. In particular,
focusing the attention on one of the six maps (e.g., the first map
from the right corresponding to p.sub.1=1670 nm) it is possible to
appreciate the excellent correspondence between the peaks of
maximum intensity enhancement predicted by the proposed
interference model and the experimentally measured peak
positions.
The excellent agreement between experiment and simulation strongly
supports the SPP interference model, thus providing for full
control and tunability of the light transmission as a function of
any of the three parameters: p.sub.1, p.sub.2 and .lamda.. In
addition, a fit of the experimental data using the SPP interference
model allows the determination of the effective SPP excitation
efficiency (.beta.) as a function of wavelength. Specifically,
.beta. decreases from .about.0.3 at 460 nm to .about.0.15 at 760
nm. Accordingly, color maps of transmitted intensity normalized to
single slit for Ag/Air and Ag/water interfaces were simulated,
using the well-known dielectric constant for silver and water.
Application of Plasmonic Interferometery to Sensing of Dielectric
Materials.
According to aspects of the present disclosure, methods of
fabrication and testing of nanohole, nanoslit, and nanogroove
arrays that enable resonant enhancements of the incident laser
intensities over a broad spectral range are provided. The same
nano-patterned structures can be used to achieve the following
simultaneously: (a) resonantly enhance the intensity of the
incident light source used to excite the propagating surface
plasmons and (b) enhance the device sensitivity to small refractive
index change (measured in terms of relative change in transmitted
intensity).
The sensitivity of the interferometer to surface changes (such as
would be the case when an analyte is present) is demonstrated by
modification of the surface using various analytes deposited on the
sensor surface, such as semiconductor (CdSe) quantum dots (QDs),
glucose molecules in solution, fluorescent dyes in solution, or
small proteins in solution. FIG. 10a shows a schematic of uncoated
interferometer consisting of a groove and a slit milled in a metal
film; upon uniform illumination of the device with a focused
Gaussian laser beam, the intensity measured through the slit
aperture results from the interference between the propagating SPP
and the incident light. As a function of separation distance D, the
light intensity transmitted through the slit of different
interferometers shows maxima and minima, corresponding to
constructive and destructive interference between the SPP and the
incident light beam. FIG. 10c shows experimental measurements of
the light intensity at 687.9 nm transmitted through the slit of
several uncoated interferometers. Each data point corresponds to
the transmission intensity through an interferometer with specific
slit-groove separation distance, normalized by the light intensity
transmitted through an identical but isolated slit aperture on the
same film. The ensemble of data is hereafter referred to as the
sensor "interferogram." The top panel of FIG. 10c shows the
transmitted intensities for uncoated interferometers (in air),
which serve as our reference interferogram. When coated with a
uniform, 20 nm-thick film of semiconductor (CdSe) quantum dots
(QDs), as schematically shown in FIG. 10b, the sensor array shows a
remarkably different interferogram (See middle panel of FIG. 10c).
The interferogram peaks and valleys shift toward lower values of D,
as a result of the enhanced refractive index. For a 10-.mu.m long
interferometer, introduction of the QDs causes a transmission
minimum to become a transmission maximum, as indicated by the
vertical blue line. The bottom panel of FIG. 10c reports the
measured change in light transmission through coated
interferometers, relative to uncoated interferometers, as a
function of groove-slit separation distance. This relative change
can be as much as 100% for a 10 .mu.m-long interferometer.
The results suggest a detection capability as low as 0.6
ng/mm.sup.2 in analyte surface mass density, corresponding to 0.1%
change in refractive index for this particular design. The
disclosed approach enables measurement not only of the real part of
the refractive index, but of the imaginary part as well, which is
related to the absorption properties of the analyte to be detected.
FIG. 10d shows the experimental interferograms for uncoated (top
panel) and QD-coated (middle panel) interferometers recorded at
514.5 nm. At this wavelength, the SPP energy is greater than the
bandgap of the CdSe QDs (designed to have an absorption edge at 600
nm). The SPP can therefore be absorbed while propagating along the
metal/dielectric interface (See schematic in FIG. 10b), causing an
exponential decrease of the transmitted intensity as a function of
separation distance D, in addition to a phase shift. The presence
of the CdSe QDs is revealed as a change in both phase and envelope
amplitude of the sensor interferogram. FIG. 10e is a schematic of
the CdSe quantum dot used to coat the surface in this series of
experiments. It includes a CdSe core and a trialkyl phosphate
surface functionalization.
The light intensity transmitted through the slit of each
interferometer can be thought of as the result of an interference
process between the incident beam (with amplitude E.sub.0) and the
propagating surface plasmon polariton (with amplitude E.sub.SPP),
whereas more than one SPP can be excited using more than one groove
as the excitation source. The phase difference between the
propagating surface plasmon polariton and the incident beam leads
to a modulated light intensity whose expression is given below. The
phase difference .delta. carries information of the analyte through
the refractive index n.sub.SPP.
.times..times..times..beta.eI.delta..times. ##EQU00002##
.times..times..times..times..beta.eI.delta. ##EQU00002.2##
.times..delta..times..phi.I.times..alpha..times. ##EQU00002.3##
.times..beta..times.e.alpha..times..times..times..beta.e.alpha..times..ti-
mes..times..function..times..phi. ##EQU00002.4##
So with the normalized transmitted intensity v. groove-slit
separation distance D at a certain wavelength, effective SPP
excitation efficiency .beta. and SPP refractive index can be
extracted by data analysis. FIG. 11a shows .beta. as a function of
wavelength in Ag/air and Ag/water interface. In addition, the
refractive index of either metal or dielectric material can be
extracted if the other constant is known. FIG. 11b shows the
dispersions of water and Ag, which agree well with the data from
references. Accordingly, the dispersion of any dielectric above the
sensing surface, i.e. the dielectric's fingerprint, can be found
through data manipulation, which makes a spectroscopic
ellipsometer. And it is also feasible to find out the composition
of the dielectric material on the surface.
Application of Plasmonic Interferometry to Glucose Sensing.
Glucose is an ideal analyte for sensing applications because (1) it
was one of the first molecules for which a biosensor was developed;
(2) 346 million people worldwide are affected by diabetes and thus,
require constant monitoring of their glucose levels; and (3) a good
number of papers report results on glucose sensing, which can serve
as a basis for comparing the performance of our plasmonic
interferometers and determining the feasibility for cytokine and
other biochemical analyte detection. Moreover, it would be ideal to
find alternative methods able to sense even lower glucose
concentrations, potentially allowing the use of saliva or tears for
noninvasive glucose sensing and real-time monitoring. It should be
noted that the principles described herein can be applied to
monitor the concentration of any organic or non-organic analyte. To
illustrate the feasibility for glucose sensing, thousands of
plasmonic interferometers were patterned onto a single biochip
equipped with a polydimethylsiloxane (PDMS) micro-channel. Various
concentration of glucose solutions in water were flowed into and
out of the microfluidic channel at a constant rate of 150 .mu.L/min
using two microsyringe pumps, and the transmitted light intensity
was monitored for each plasmonic interferometer. The continuous
flow together with the extremely low power density allowed the
temperature of the device to be held constant throughout the
sensing experiment. The microfluidic system can be part of a sample
holder system. According to aspects of the disclosure, the sample
holder can further include inlet and outlets positioned to permit
inflow and outflow of a second fluid in the sample holder.
A compact, high-throughput plasmonic sensor based on surface
plasmon interferometry optimized for real-time monitoring of
glucose in aqueous solutions consists of a spatially dense, planar
array of plasmonic interferometers (>1,000/mm.sup.2), where each
interferometer is composed of 100 nm-wide, 10 .mu.m-long grooves
flanking a 200 nm-wide slit etched in a 300 nm-thick silver film
using focused ion-beam milling. The distances between each groove
and the slit were varied between 0.25 to 10 .mu.m in steps of 25
nm. The detection limit of the plasmonic sensor for glucose in
aqueous solutions is 5.5 .mu.M with a sensitivity of 105,000%/RIU
(refractive index units) at 590 nm. Based on results shown in FIG.
5, the device with p.sub.1=0.57 .mu.m and p.sub.2=5.70 .mu.m was
chosen to perform the glucose sensing experiment. As discussed
above, FIG. 12a illustrates the normalized per-slit transmitted
intensity spectra of this device, measured as a function of
wavelength for increasing concentrations of an aqueous glucose
solution. A wavelength shift (.DELTA..lamda.) is observed at all
incident wavelengths. Increasing glucose concentration enhances the
refractive index of water (the dielectric material), and results in
a red-shifted interference spectrum.
FIG. 12(a) shows the normalized per-slit transmission spectra of a
groove-slit-groove plasmonic interferometer with p.sub.1=0.57 .mu.m
and p.sub.2=5.70 .mu.m, measured at various concentrations of
glucose in water. A wavelength shift (.DELTA..lamda.) is observed
at all wavelengths. Increasing glucose concentration enhances the
refractive index on the surface, thus resulting in the observed
red-shift in the normalized transmission spectra.
In contrast to existing approaches that use single excitation
wavelength, the proposed device operates in a broad wavelength
range, allowing a polychromatic detection of chemical analytes. By
correlating the wavelength shift with the known concentration, a
calibration curve can be determined for each device.
FIG. 12(b) shows another figure of merit used to characterize our
device response, i.e. the relative intensity change
(.DELTA.I/I.sub.0) normalized to pure water for the same device, at
various concentrations of glucose in water. Some wavelengths do not
show any significant change in transmitted light intensity, forming
"nodes" that are characteristic of each specific device. For this
particular device, the maximum relative intensity change is
achieved at a wavelength of 610 nm, with values of up to 40%. In
summary, by monitoring the .DELTA..lamda. and .DELTA.I/I.sub.0 at
all wavelengths and for various glucose concentrations, we were
able to sense glucose concentrations in a broad range from
0.1-20,000 mg/dL corresponding to a change in refractive index
units (RIU) of .about.0.014.
The relative intensity change (.DELTA.I/I.sub.0) is given by
.DELTA..times..times..times..times. ##EQU00003##
where I.sub.glucose is the transmitted light intensity through the
slit of a plasmonic interferometer at a specific glucose
concentration, and I.sub.0=I.sub.warer is the reference transmitted
intensity through the slit of the same interferometer in pure water
(i.e., zero glucose concentration).
FIG. 13 shows the performance of the same device analyzed by
plotting the experimental and simulated values for .DELTA..lamda.
and .DELTA.I/I.sub.0 as a function of glucose concentration. The
experimental (symbols) and simulated (dash line) .DELTA..lamda. at
a center wavelength of 610 nm and .DELTA.I/I.sub.0 at 590 nm (gray
line and circles) and at 610 nm (black line and squares) are shown
in FIG. 13, panels a and b, respectively. The simulated curves are
in excellent agreement with the experimental data. .DELTA..lamda.
and .DELTA.I/I.sub.0 show significant variation as a function of
glucose concentration, and can therefore be used to infer the
concentration of glucose in solution. Furthermore, as shown in FIG.
13(b), it is evident that at close but different wavelengths, the
device response can be remarkably different, with higher
sensitivity observed at 610 nm.
FIG. 14 shows a sensing performance comparison at 590 nm for four
devices, three of which have a constant p.sub.1=0.57 .mu.m and
varying p.sub.2=1.85, 5.70, and 9.75 .mu.m, and the fourth with
p.sub.1=5.70 .mu.m and p.sub.2=9.75 .mu.m. Panels a and c in FIG.
14 describe the wavelength shift, and panels b and d report the
relative intensity change as a function of glucose concentrations,
spanning 5 orders of magnitude, which correspond to a total
refractive index change of only .DELTA..sub.n=0.02. The gray boxes
in FIG. 14b highlight the physiological concentration ranges of
glucose in saliva (lighter gray area) and serum (darker gray area),
respectively. Typical physiological concentrations range between
0.36 and 4.3 mg/dL in saliva and 50 and 144 mg/dL in serum. The
inset to FIG. 14a shows representative relative intensity change
spectra for a device with p.sub.1=0.57 .mu.m and p.sub.2=9.75 .mu.m
measured at four glucose concentrations. By monitoring the changes
in the experimental spectra taken at different glucose
concentrations, scattered data of .DELTA..lamda. and
.DELTA.I/I.sub.0 can be obtained, as plotted in FIG. 14 for a
wavelength of 590 nm. When panels a and b in FIG. 14 are compared,
it is observed that, at low glucose concentrations (0.1-500 mg/dL),
.DELTA..lamda. does not show a significant change, whereas
.DELTA.I/I.sub.0 is a more reliable parameter for detection of
glucose. At higher glucose concentrations (500-14 000 mg/dL) both
.DELTA..lamda. and .DELTA.I/I.sub.0 can be used to detect the
presence of glucose and quantify its concentration. The dashed
lines are calibration curves obtained by least-squares fittings of
the scattered experimental data points. The proposed plasmonic
interferometers are able to sense the lowest glucose concentrations
typically found in saliva.
At all concentrations, .DELTA.I/I.sub.0 seems to outperform
.DELTA..lamda.. In order to better quantify the device sensitivity,
figures of merit can be calculated as the slopes of the calibration
lines obtained by fitting .DELTA..lamda. and .DELTA.I/I.sub.0 data
as a function of concentration. In particular, by dividing the
wavelength shift .DELTA..lamda. by the difference in the refractive
index (.DELTA.n) between two glucose solutions we can define a
figure of merit relative to the wavelength shift (FOM.sub..lamda.)
at each given concentration, i.e.
.lamda..DELTA..times..times..fwdarw..times..DELTA..lamda..DELTA..times..t-
imes. ##EQU00004##
Similarly, dividing the difference in the relative intensity change
.DELTA.I/I.sub.0 by .DELTA.n, the figure of merit relative to the
intensity change (FOM.sub.I) can be derived, at each
concentration
.DELTA..times..times..fwdarw..times..DELTA..times..times..DELTA..times..t-
imes. ##EQU00005##
The device with p.sub.1=0.57 .mu.m and p.sub.2=9.75 .mu.m
(triangles) shows a FOM.sub.I of .about.166 000%/RIU in the glucose
concentration range typically found in saliva (0.2-8 mg/dL) and
.about.10 000%/RIU in the concentration range typically found in
blood serum (40-400 mg/dL). For the device with longer p.sub.1=5.70
.mu.m (orange circles), the measured FOM.sub.I is even higher,
reaching .about.884 000 and .about.17 000%/RIU in the glucose
concentration ranges for saliva and serum, respectively. This
device is able to detect a refractive index change as low as
3.times.10.sup.-7 RIU. Experimentally, the relative intensity
change is found to be a better figure of merit for detection of low
glucose concentrations. The data suggest that at the lowest glucose
concentrations, adsorption of glucose molecules directly onto the
metal surface of the plasmonic interferometer determines an
increased glucose concentration right at the device surface and
higher FOM.sub.I than those simulated using a uniform glucose
concentration in solution. Functionalization of the sample surface
with linkers specific to glucose can further increase the device
sensitivity and specificity. At higher glucose concentrations
(500-14 000 mg/dL), FOM.sub..lamda. is between 370 and 630 nm/RIU
for the three devices (FIG. 14c). The device with p.sub.1=0.57
.mu.m and p.sub.2=9.75 .mu.m, shows a decrease in FOM.sub.I from
.about.16 000%/RIU (FIG. 14b) to .about.1000%/RIU (FIG. 14d), in
agreement with the trend observed in the simulations, showing that
plasmonic interferometers optimized for detection of low refractive
index changes are indeed characterized by higher figures of merit
at lower glucose concentrations. The measured figures of merit are
at least 1 order of magnitude greater than what is reported in the
literature, opening up the possibility to use plasmonic
interferometers for detection of low concentrations of clinically
relevant molecules. Moreover, the typical sensing volume of a
plasmonic interferometer is only 20 fL, which corresponds to a
sensed mass of 0.02 fg or .about.67 000 molecules. If desired,
sensing specificity can be achieved for multiple analytes by
functionalizing the surface with antibodies that have high affinity
to specific analytes.
Typical physiological concentrations range between 50-144 mg/dL in
serum and between 0.36-4.3 mg/dL in saliva. The disclosed plasmonic
interferometers are able to sense the lowest glucose concentrations
typically found in saliva. Experimentally, a maximum sensitivity of
.about.32,000 nm/RIU and relative intensity change of
.about.105,000%/RIU were observed. These numbers are greater than
those found in the literature (See Table 2 below). One proposed
device according to aspects of the disclosure is able to detect as
low as 3.times.10-7 RIU, and it uses sensing volumes as small as 5
fL. These results demonstrate that plasmonic interferometers are
candidates for the development of alternative optical sensors that
can sense mass down to 0.02 fg, or .about.67,000 molecules. The
disclosed devices can therefore be used for the development of a
minimal, non-invasive glucose measurement technique in saliva,
which can greatly improve the lifestyle of diabetic patients.
Various experimental results suggest the possibility to detect
analyte mass as low as 20 femtograms, by using various figures of
merit, such as wavelength shift in the interference spectra and
relative intensity change caused by a change in refractive index,
determined by an increase in glucose concentration in solution.
Plasmonic Interferometry Coupled to Dye Chemistry for Specificity
and Enhanced Sensitivity.
According to aspects of the present invention, the sensor
selectivity to glucose is improved using a proposed novel molecular
recognition scheme that couples plasmonic interferometry with dye
chemistry, specifically using 10-acetyl-3,7-dihydroxyphenoxazine
(Amplex Red). According to aspects of the present invention,
glucose oxidase is added in solution to rapidly convert D-glucose
into D-gluconolactone and H.sub.2O.sub.2 in a 1:1 stoichiometry
H.sub.2O.sub.2 reacts with horseradish peroxidase (HRP) to oxidize
Amplex Red into resorufin, a dye molecule which is characterized by
a strong optical absorption coefficient at .about.571 nm (see FIG.
15). The reaction can be monitored in real-time by simply measuring
changes in the light intensity transmitted through the slit of each
interferometer. As a result of the increased concentration of
resorufin, the solution's refractive index and the absorption
coefficient will increase, resulting in, respectively (1) a
spectral shift in the interference conditions (.DELTA..lamda.), and
(2) an intensity decrease due to increased SPP absorption
(.DELTA.I). These two changes, (.DELTA..lamda. and .DELTA.I) will
provide information on the change in refractive index which can be
directly correlated to the glucose concentration. The presence of
.beta.-D-Glucose in a reaction cocktail of glucose oxidase,
horseradish peroxidase, and Amplex Red results in a red solution
and in high absorbance that peaks at .lamda.=572 nm.
According to aspects of the present invention, plasmonic
interferometers are used for monitoring the kinetics of Amplex Red,
glucose oxidase and glucose reaction. The effects of absorption
using Amplex Red with a maximum absorbance at 572 nm on the
spectral response of the plasmonic interferometer is shown in FIG.
16a. The absorption of the dye is directly proportional to the
concentration of glucose above the sensing surface, as observed by
the decrease in the relative intensity change between 500-600 nm.
At wavelengths higher than 600 nm, there is a small wavelength
shift due to the refractive index change caused by the increasing
glucose concentration. The overall sensitivity is also observably
higher than the plasmonic interferometer without dye present as
seen in FIG. 16b. This approach demonstrates for the first time the
enhanced specificity and selectivity of coupling the plasmonic
interferometer with Amplex Red allowing for the development of
point-of-care diagnostic tools for biomedical sensing of glucose
and other clinically relevant analytes. The inventive matter
involves the combining of plasmonic interferometry with dye
chemistry as described. While certain specifics compounds are noted
above (e.g. using glucose and 10-acetyl-3,7-dihydroxyphenoxazine
(Amplex Red)), the general idea includes the use of dye chemistry
to alter a molecular target as to optical absorption coefficient,
refractive index and related optical characteristics.
Application of Plasmonic Interferometry to Trauma.
A plasmonic-based device that accurately measures, in real-time,
the cytokine levels in polytrauma patients is provided. The demand
for clinical testing of cytokine levels in serum has increased
significantly because of their strong correlation to predicting the
clinical course and outcome of trauma patients. This demand arises
from the fact that a physician must make a decision on the timing
and type of surgical treatment for a poly-trauma patient in the
absence of real-time data. As such, these decisions are susceptible
to human error, which can lead to systemic complications such as
multi-organ dysfunction syndrome (MODS) and death of the patient.
The physiological reaction to injury is most notably an
inflammatory response that involves an interconnected network of
protein mediators (cytokines, chemokines, nitric oxide) and
effector cells (neutrophil, monocytes/macrophages and endothelial
cells).
A controlled local inflammatory response is beneficial during the
acute period following major trauma as the endogenous response
systems modifies signaling pathways to limit further injury,
prevent infection and initiate healing. When an exaggerated local
inflammatory response propagates systemically, however, the result
is known as a systemic inflammatory response syndrome (SIRS), which
can lead to acute respiratory distress syndrome (ARDS), MODS and
ultimately death.
In addition to initial injuries, long surgical procedures and
fixation of fractures performed immediately after trauma induce
surgical stress and can increase the risk of ARDS/MODS. About two
decades ago, it was believed that surgical stabilization of all
fractures had to be performed within 24 hours after injury.
However, this paradigm is under heavy scrutiny as new technologies
enable us to further elucidate the mechanism behind the development
of ARDS/MODS.
The "second-hit" phenomenon theory suggests that a patient in a
hyper-inflammatory physiologic state following an initial injury
(first hit) may be pushed over a threshold for the development of
MODS by an ill-timed surgical procedure (second hit). Surgeons have
noted that a focus on resolving hemorrhage and stabilizing
high-priority injuries in the first 72 hours, and thereby delaying
extensive orthopedic procedures, can greatly improve the chance of
patient survival. The approach of staging surgical intervention is
known as damage control orthopedics (DCO). In order for DCO to gain
widespread implementation, a standardized set of guidelines to base
clinical decisions regarding the appropriate timing and extent of
surgical intervention in a poly-trauma patient is required.
One method for differentiating those patients who are ready and can
tolerate prolonged surgical procedures is to monitor their cytokine
response. The systemic release of inflammatory cytokines such as
TNF-.alpha. (Tumor Necrosis Factor-alpha, involved in the acute
phase response), or the interleukins IL-1, IL-6 and IL-8 is
proportional to the severity of trauma, surgical stress response
and sepsis. Elevated IL-6 and IL-8 levels have been found to
increase significantly in patients in hemorrhagic shock and in 95%
of patients undergoing routine surgical procedures. The major
limitation in the accuracy of measuring cytokine levels lies in
their inherent short half-lives ranging from approximately 6
minutes for IL-1 and 20 minutes for TNF-.alpha..
Currently, cytokine levels in blood serum is predominantly
quantified by immunoassay such as commercially available
enzyme-linked immunosorbent assays (ELISA), radioimmunoassay and
immunoaffinity column assays. Common amongst these laboratory
techniques is the need for incubation (2-3 hours) and the
subsequent delay in the diagnosis. Recently, optical sensors based
on Surface Plasmon Resonance (SPR) technology such as the
Biacore.RTM. have been used to monitor cytokine concentrations in
real time and without labeling. However, these machines are bulky,
expensive and require highly qualified personnel to operate.
In one aspect, a device capable of quantifying markers of
inflammation in serum in real-time is described. The device
provides physicians with the ability to assess the type and timing
of surgery that would reduce the impact of the second hit insult.
The device based on novel plasmonic architecture can accurately and
rapidly measure the level of cytokines in blood serum in real-time.
Cytokine levels reflect the severity of injury in a patient. The
disclosed invention provides a new method for measuring cytokine
levels in patients suffering from traumatic injury (e.g., American
soldiers, victims of vehicular impact).
Under aspects of the present disclosure, the provided inventive
plasmonic interferometer allows for multiplex, real-time
quantification of cytokines in addition to surmounting all of the
afore-mentioned limitations. The sensitive biological element
consists of cytokines in blood serum, delivered using micro-fluidic
techniques.
According to alternative aspects of the present disclosure, methods
of device fabrication and optimization for effective cytokine
detection in blood serum are provided.
According to alternative aspects of the present disclosure, methods
of development and implementation of surface chemistry by which to
attach selective sensing elements for targeted cytokines are
provided. Table 1 lists major cytokines involved in trauma.
TABLE-US-00001 TABLE 1 Major inflammatory cytokines involved in
trauma..sup.1 Molecular Major cytokines Mass The role of cytokines
Tumor necrosis factor-.alpha. 17.5 kDa Potential marker of
(TNF-.alpha.) inflammation.sup.[a] Interleukin-1 (IL-1) 18 kDa
Elaboration and release of other cytokines during immune
response.sup.[b] Interleukin-6 (IL-6) 20.3 kDa Reliable marker of
the severity of injury, magnitude of systemic inflammation, and
mortality rate.sup.[c] Interleukin-8 (IL-8) 8.3 kDa Determinant of
postinjury mortality in pediatric blunt- trauma patients.sup.[d]
Interleukin-10 (IL-10) 18.8 kDa Down-regulation of pro-
inflammatory cytokines.sup.[e] .sup.1References: .sup.[a]Giannoudis
P V, Injury 34, 397-404 (2003); .sup.[b]Sutton C, The Journal of
Experimental Medicine 203, 1685-1691 (2006); .sup.[c]Mimasaka S,
Injury 38, 1047-1051 (2007); Hack C E, Blood 74, 1704-1710 (1989);
Gebhard F, Arch Surg 135, 291-295 (2000); .sup.[d]Ozturk H, Pediatr
Surg Int 24, 235-239 (2008); .sup.[e]Pastores S M, Acad Emerg Med
3, 611-622 (1996).
According to alternative aspects of the present disclosure, a
plasmonic interferometer used for the rapid, accurate, and low-cost
measurement of cytokines can significantly improve the outcome of
triage at an emergency center. The real-time sensing capability of
the plasmonic interferometer can also help elucidate pro- and
anti-inflammatory release mechanisms needed to verify the second
hit phenomenon. Our plasmonic interferometers have the potential to
become an invaluable point-of-care diagnostic tool for orthopedics
surgeons to monitor the evolution of clinical cytokines and guide
them in making informed decisions to treat and operate on patients
affected by polytrauma, preventing the onset of MODS.
Immobilization of Antibodies and Capture Proteins Specific to
Targeted Cytokines and Other Biochemical Analytes
Under alternative aspects of the present disclosure, chemistry
methods are provided for optimally modifying the surfaces of the
plasmonic interferometers. For example, FIG. 17 shows a schematic
for human anti-IL6 (hAnti-IL6) or other capture proteins
immobilized onto Au (Ag) substrates using EDC/NHS coupling.
As a starting point, substrates were prepared from clean quartz
measuring one square inch. An adhesive layer (4 nm) of either
titanium (Ti) or chromium (Cr) was e-beam deposited onto the quartz
followed by e-beam deposition of a thin layer (300 nm) of either
gold (Au) or silver (Ag). The resulting metal-coated quartz was
immersed in an ethanol solution containing 1 mM
11-mercaptoundecanoic acid (11-MUA) and incubated overnight at room
temperature. After rinsing the substrate with copious amounts of
ethanol followed by acetone, the substrate was immersed in an
aqueous solution containing 5 mM
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 5 mM
N-hydroxysuccinimide (NHS) for 4 h to generate a reactive ester at
the terminus of the self-assembled monolayer. The substrate was
rinsed with de-ionized water to remove any unreacted EDC or NHS
prior to its exposure to an aqueous solution containing hAnti-IL6
(8 .mu.g/mL) for 18 h. The ensuing chemical reaction insures that
IL-6 is covalently attached to the substrate surface via amide bond
formation between the primary amine group on IL-6 and the reactive
ester of 11-MUA. Prior to FTIR analysis, the substrate was rinsed
with de-ionized water and dried under nitrogen.
Surface modification on a gold-coated quartz substrate was
performed and infrared spectra of the immobilization process of
hAnti-IL6 were collected on a Nicolet Nexus 670 FTIR spectrometer.
FIG. 18a shows sequential infrared spectra of hAnti-IL6 immobilized
onto gold-coated quartz substrate via EDC/NHS coupling chemistry.
The specular reflectance was measured and converted to absorbance.
Seven characteristic absorbance peaks are labeled with bold numbers
in FIG. 18a: (1) asymmetric stretch of CH.sub.2 at 2950 cm.sup.-1;
(2) NHS esters stretching mode at 1815 cm.sup.-1; (3) symmetric
stretch of NHS carbonyls at 1750 cm.sup.-1; (4) symmetric stretch
of NHS carbonyls at 1740 cm.sup.-1; (5) carbonyl stretching of the
carboxylic acid groups (--COOH) of 11-MUA at 1710 cm.sup.-1; (6)
carbonyl stretching mode corresponding to the amide bonds (amide I)
in hAnti-IL6 at 1675 cm.sup.-1; and (7) a combination of N--H
bending and C--N stretching modes of the amide bonds (amide II) in
hAnti-IL6 at 1550 cm.sup.-1. The amide I and amide II peaks (see
FIG. 18a: peaks 6 and 7, blue line) are indicative that hAnti-IL6
was immobilized onto the gold-coated quartz substrate. The same
immobilization scheme can be applied for the immobilization of
other capture proteins such as glucose oxidase (GOx) as seen in
FIG. 18b.
FIG. 18a shows evidence of surface functionalization using
hAnti-IL6 immobilization on a metal surface. Increasing the
immobilization time leads to an increase in the surface coverage
(FIG. 18a); the measurements evidence a continuous, uniform
monolayer of hAnti-IL6 covalently bound at the surface.
Under alternative aspects of the present disclosure similar
procedures can modify plasmonic interferometers with the other
targeted cytokines and other analytes of interest. Further
characterization of the modified surfaces can include antibody
staining, contact angle measurements, and collection of X-ray
photoelectron spectra to insure the coatings are homogeneous over
the entire surface of the substrate.
Under alternative aspects of the present disclosure, protection of
the metal surface (in particular Ag) from oxidation from
biochemical reagents and buffers, a thin film of aluminum oxide
(Al.sub.2O.sub.3), silicon dioxide (SiO.sub.2) and the like can be
deposited on top of the metal surface. This causes the surface to
become more hydrophilic in the case of Ag and Al.sub.2O.sub.3 (see
FIG. 19a). Furthermore, this method enables the chip to be reused
subsequent to chemical dye or other reagent exposure, as indicated
by the return of the peak positions at the same wavelengths
observed in FIG. 19b.
Device Integration and Prototype Designs.
FIG. 20 shows an implementation of ten 1 .mu.m-long interferometers
in an area of 77 .mu.m.sup.2, demonstrating an array density of 13
million devices per square centimeters. The individual
interferometers are arranged in columns and are spaced in a way to
minimize the surface plasmon interference between neighboring
interferometers of adjacent columns.
Fabrication of several plasmonic interferometers on the same chip
enable fast characterization of the optical response as a function
of the above mentioned geometric parameters, and further can allow
for a comparison with simulation results enabling high-throughput
experiments.
FIG. 21a shows how the skin depth and propagation length of surface
plasmons vary for different wavelengths, for two fluids: water and
air. The propagation length is defined as the distance that a
surface plasmon should propagate for its intensity to decay by a
factor of 1/e. For example, in FIG. 21b, at .lamda.=500 nm, the
propagation length is about 3 .mu.m. Between two neighboring
interferometers, a separation distance equal to two propagation
lengths adequately provides sufficient isolation between the two
neighboring interferometers. For each propagation length there is
an exponential decay of the surface plasmon intensity. Other ways
to provide optical isolation between neighboring interferometers
can include, for example, deposition of platinum or chromium.
Platinum and chromium have strong absorption characteristics,
therefore surface plasmons lose their intensity when they propagate
through them. Platinum and chromium can be deposed using standard
thin-film deposition techniques. The thin film can be as long as
the slit of the neighboring interferometer and have a width, for
example, of 1 .mu.m. The thickness of the thin film can vary
between 2 nm and 3 nm.
Under alternative aspects of the present disclosure, a hand-held
device is provided. The hand-held device can be configured with a
microarray of plasmonic interferometers with surfaces chemically
modified to capture specific analytes found in bodily fluids. For
example, a sub-microliter sample of serum can be introduced to the
interferometer chamber through capillary action. Subsequent to
photonic detection, the data can be displayed, for example, on a
LCD display. Specific recognition of targeted molecules eliminates
the need for extensive extraction and/or purification of the
sample. Additionally, the integration of the microarray detector
with MEMS technology can enable the construction of a biochemical
photonic sensor that can be mass produced at low cost.
Under alternative aspects of the present disclose, typical
interferometer dimension and separation distances can be scaled
down by use of nano-imprint lithography in order to fabricate and
integrate as many as one million interferometers per centimeter
squared on a metal film for improved sensor throughput, which would
be desirable, for example, for faster drug screening and discovery.
A device according to aspects of the present disclosure is shown
schematically in FIG. 22. A sub-microliter sample of analyte in
solution can be introduced to the interferometer chamber through
capillary action (not shown) comprising inlet and outlets for
positioned to permit inflow and outflow of a second fluid in the
sample holder. By varying the normal incident wavelength, each CCD
pixel can record the normalized transmission spectrum through an
individual plasmonic interferometer. Flow of the analyte in
solution and further binding to the specific linker will produce a
shift in the spectrum. Subsequent analysis in situ will determine
calibration curves. Therefore, the proposed sensor can detect the
refractive index change at various wavelengths simultaneously.
Additionally, the CCD camera integrated on the back of our sensor
chip will allow multiplexed, real-time detection of light
transmitted through each and every plasmonic interferometer, thus
enabling individual addressability and multispectral imaging
capabilities to collect 2D maps in a single reading. A CMOS sensor
can alternatively be used to record the light through each
individual plasmonic interferometer.
This can result to high-throughput measurements of concentration
and type of analytes (selected according to specific linkers) for
monitoring several patients simultaneously. For example, by
employing linkers specific to different analytes, and using 1,000
plasmonic interferometers in parallel, a multiplexed screening of
various biochemical analytes for 30+ patients can be accomplished
in one run.
FIG. 23a shows a prototype schematic of a glucose monitor,
according to aspects of the present disclosure. FIG. 23b shows an
actual photograph of the prototype 2300. The prototype consists of
a biochip 2301, an integrated plasmonic sensor array chip 2302, an
LCD display 2303, a microfluidic channel 2304, and an integrated
light source. The prototype also consists of an optical meter,
which is a point-of-care, battery-powered, cube-shaped device that
opens and closes with a flex-hinge. When opened, the top panel is a
LCD screen that serves both as a display panel and a light source
used for optical sensing. The bottom panel contains a hidden CCD
camera located directly underneath a recess for which the biochip
can be placed. The camera is responsible for recording the
intensity change for each interferometer at its corresponding pixel
locations. The biochip comprises a plurality of plasmonic
interferometers and can be packaged individually.
In other embodiments, a kit is provided for operation of a glucose
test. The kit includes chemicals necessary for reaction with saliva
sample for generation of dye indicator, e.g., horseradish
peroxidase, glucose oxidase and 10-acetyl-3,7-dihydroxyphenoxazine
(Amplex Red) and instructions for use in the detection of glucose
in saliva using a plasmonic interferometer device. FIG. 24 is a
graphical illustration of the instructions to operate the testing
glucose monitor.
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